Carcinogenesis is a multistep process involving heritable alterations. There is evidence for the involvement of two distinct classes of genes in carcinogenesis proto-oncogenes and tumor suppressor genes. The heritable alterations that have been identified in tumors include point mutations, gene deletions, gene amplifications, gene rearrangements, numerical chromosome changes and modifications in DNA methylation patterns. Mutations at the tk locus in the L5178Y mouse lymphoma cell can be considered a cellular model for alterations in tumor suppressor genes in that the tk locus is heterozygous as is the tumor suppressor gene and in that all the alterations except for modifications of DNA methylation patterns have been identified in both systems. In order to determine if changes in gene expression due to changes in methylation patterns can cause mutation. we treated mammalian cells with 5-azacytidlne and other cytidine analogs and found that their ability to induce differentiation correlated with these compounds' ability to Induce trifluorothymidine resistance. We have isolated these TFTr cells and are examining them for the genetic lesions that cause the mutagenic phenotype. The adduction of DNA by a particular carcinogen's heterogeneous both structurally and functionally. "Hotspots" in both the adduction and its repair can occur because of primary sequence effects, effects of local chromatin structure, the binding of proteins to promoters and enhancer sequences, and the transcriptional and methylation states of the gene. By examining adduction and repair at the base-pair level, it should be possible to address some of these above factors as well as determine how these factors affect repair. Also, by comparing the adduction and mutational spectra at a specific gene, it will be possible to identify hotspots. We are in the process of improving a gene technique called ligation-mediated polymerase chain reaction to allow us to examine the adduction and repair of genomic DNA at individual base pairs within a gene.